IDENTIFICATION OF AN E3 LIGASE REGULATING THE CATALYTIC SUBUNIT OF RNA POLYMERASE I

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) into a 47S rRNA pre-cursor; this is the first and rate-limiting step of ribosome biogenesis. Cancer cells possess abnormally high rates of ribosome biogenesis to match their increased proliferative and biosynthetic activities. Many oncogenic drivers achieve this through the constitutive activation of Pol I transcription, making Pol I a promising target for cancer therapeutics. Current studies focus on the regulation of Pol I activity through the modification of its transcription factors and the rDNA, but very little is known about the stability or regulation of the enzyme itself. The largest subunits of Pols II and III are degraded by the ubiquitin proteasome system upon transcription arrest. The stress-induced degradation of the enzyme could thus be an evolutionarily significant means of regulating RNA polymerase activity. We recently discovered a first-in-class small molecule, BMH-21, that inhibits Pol I transcription and induces the degradation of RPA194, the largest and catalytic subunit of Pol I. Moreover, the inducible degradation of RPA194 occurs predominantly in cancer cells and correlates with cancer cell death. We have found that RPA194 degradation occurs through the ubiquitin proteasome system, drawing parallels to prior studies of the proteasome-mediated degradation of large RNA polymerase subunits. The aim of this thesis was to study the mechanisms of BMH-21-mediated RPA194 degradation. A detailed understanding of the degradation process will provide fundamental knowledge about the stability and regulation of the Pol I enzyme complex. This knowledge will also benefit therapeutic implementation of Pol I inhibitory strategies. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes associated with RPA194 degradation. This screen identified a Skp-Cullin-F-box protein complex (SCF complex) as a promising candidate E3 ligase. We focused on validating the role of the identified F-box protein, FBXL14, in RPA194 degradation. To do this, we conducted knockout, knockdown, and overexpression analyses of FBXL14 in melanoma, osteosarcoma, breast cancer, and chronic myelogenous leukemia cancer cell lines. We show that FBXL14 interacts with and promotes the ubiquitination and degradation of RPA194. Further, we found that FBXL14 overexpression increased the sensitivity to BMH-21 in certain cancer cell lines and activated RPA194 turnover in degradation-resistant cell lines. We used transcriptional and chromatin engagement assays to demonstrate that the drug-induced degradation occurs after polymerase detachment from the chromatin. The Pol I inhibitors discovered in our studies have been unique in their capacity to induce the enzyme turnover. Here, we demonstrate that FBXL14 overexpression destabilizes RPA194 not only by BMH-21 treatment, but also after treatment with other Pol I inhibitory agents, suggesting that FBXL14 has a rate-limiting role in the control of the enzyme stability during transcription stress. In this study, we identified the first E3 ligase targeting the Pol I complex and developed a more comprehensive model of RPA194 degradation by Pol I inhibitors. These findings provide fundamental knowledge about the stability and regulation of the Pol I complex relevant for the mechanistic understanding of the enzyme function and the development of Pol I-targeting agents